Use of thymosin alpha 1 for preparing a medicament for the treatment of stage IV malignant melanoma

ABSTRACT

It is described the use of thymosin alpha in combination with dacarbazine and optionally with Interferon alpha, for preparing a medicament for the treatment of malignant melanoma on stage IV characterized by distant unresectable metastases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/734,592, filed Apr. 12, 2007, which is a continuation-in-part of U.S.application Ser. No. 11/424,475, filed Jun. 15, 2006, the disclosure ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the use of thymosin alpha 1 incombination with dacarbazine and optionally Interferon alpha, forpreparing a medicament for the treatment of malignant melanoma on stageIV.

Melanoma is a malignant tumor of melanocytes, which are cells derivedfrom the neural crest.

Melanomas are found primarily in normal areas of the skin, but may alsooccur in other mucosal surfaces.

Skin nevi may be suspected of undergoing malignant changes if theyappear darker or have variable discoloration, or there is itching, anincrease in size, or development of satellites.

Melanoma is unusual in that it is far more likely to metastasize thanother types of cancer and can spread to regional or distant lymph nodes,or to any of the major organ systems of the body.

The most common sites of metastasis other than the skin are the lung,liver, brain, and lymph nodes.

The clinical presentation of stage IV malignant melanoma (“high-riskmelanoma” or “H-RM”) will vary depending on the stage and site(s) ofsystemic involvement.

Melanoma occurs more frequently in males and is found in adults of allages.

The American Cancer Society (“ACS”) estimated the number of new cases ofall skin melanomas for 2005 at 59,580 and the number of deaths at 7,770.

HR-M accounts for approximately 22% of all cutaneous malignant melanomacases and is associated with a high mortality rate.

Several risk factors have been identified for melanoma. It has long beenbelieved that exposure to sunlight (i.e., ultraviolet radiation) is theprimary etiological factor in the development of melanoma, which isconsistent with higher incidence rates in populations with lessphotoprotective melanin that live closer to the equator.

Other known risk factors for melanoma include: genetics, where 5-10% ofmelanoma patients have a family history of the disease;dysplastic/atypical nevi; complexion (fair-skinned, red-headed or blondindividuals, and individuals with a high tendency to freckle are athigher risk for developing melanoma); and history of severe blisteringsunburn.

Patients diagnosed with H-RM have a strikingly worse prognosis thanpatients whose tumor are of minimal thickness/invasion and are locallyconfined.

A number of key clinical factors have been identified as prognosticindicators for melanoma, including: age; sex; characteristics of theprimary tumor (e.g., anatomic location, size, Clark's level, Breslow'sthickness, histopathological type, ulceration, inflammatory reaction);and lymph node involvement.

H-RM is generally a fatal disease due to the absence of adequatetherapeutic options.

H-RM is characterized by tumors of the skin that metastasize tovirtually every organ. The clinical presentation of H-RM variesaccording to the stage and site(s) of systemic involvement.

Early stage malignant melanoma without metastasis is treated by widefield surgical excision and has a high cure rate. While regional lymphnode removal in addition to wide field surgical excision of the primarytumor may be successful in Stage III malignant melanoma.

In stage IV malignant melanoma, characterized by distant unresectablemetastases, there is no currently available treatment. Once themetastatic process has started, the tumor becomes increasingly resistantto current methods of therapy.

Thymosin alpha 1 is a compound well known in the medical field.

Subcutaneous administration of 1 or 10 mg per day of thymosin alpha 1 tonude mice previously inoculated with human non-small cell lung cancer(“NSCLC”) cells significantly decreased tumor volume.

Pulmonary metastases in mice with methylcholanthrene-inducedfibrosarcoma were also reduced by thymosin alpha 1, and local sarcomagrowth as well as liver and lung metastases of lymphosarcoma cells weresignificantly reduced in BALB/c mice treated with thymosin alpha 1.

In Int. J. Immunopharmacol. 2000; 22:1067-76 two experiences arereported:

1) The use of Dacarbazine (DTIC) (850 mg/m² i.v. on day 1)+thymosinalpha 1 (2 mg s.c. on days 4-7) in combination with interleukin-2 (18MU/m² i.v. on days 8-12). Each cycle lasted 21 days.

2) The use of DTIC (200 mg/m² i.v. on days 1-4)+thymosin alpha 1 (1 mgs.c. on days 8-11 and 15-18) in combination with interferon alpha (3 MIUi.m. on days 11 and 18). Each cycle lasted 28 days.

These experiences showed that these treatments enhance the host immuneresponse in patients with H-RM and prolong their survival.

Annals of Oncology. 1994; 5:741-46, relates to the use of dacarbazine(850 mg/m² i.v. on day 1) in combination with thymosin alpha 1 (2 mgs.c. on days 4-7) and IL-2 (18 MIU i.v. on days 8-12) in patients withH-RM. Each cycle lasted 21 days.

Favalli (1993; Combination Therapy in Malignant Melanoma. ThirdInternational Symposium on Combination Therapies, Houston, Tex.:Institute for Advance Studies in Immunology & Aging) teaches about theuse of thymosin alpha 1 (1 mg s.c. on days 8-11 and 15-18) incombination with dacarbazine (200 mg/m² i.v. on days 1-4) and IFN-α (3MIU i.m. on days 11 and 18) in patients with malignant melanoma. Eachcycle lasted 28 days.

Current development of alternative therapies for H-RM is directed towardimmunotherapies. Adjuvant immunotherapy agents designed to augment theimmune response are under development and include melanoma vaccines,interferons (“IFNs”), interleukin-2 (“IL-2”), and tumor-infiltratinglymphocytes, and plasmid-based DNA vaccines.

Trials are being conducted to evaluate alternative immunotherapy agentsin patients with H-RM have generally yielded less than encouragingresults (Cancer Inves. 23:323-37; 2005). In general, large randomizedtrials have not provided any evidence of significant clinical benefit,despite the initial promising results.

While the annual incidence of malignant melanoma is on the rise,long-term studies demonstrate that current therapeutic options, formalignant melanoma on stage IV characterized by distant unresectablemetastases, only produce limited results with little impact on thepatient's overall survival.

Trials conducted with the interferons and interleukins in combinationwith dacarbazine have not demonstrated a clinical advantage overdecarbazine monotherapy in advanced melanoma. Immunotherapeutic agentsin combination with lymphokine-activated lymphocytes have not been foundto improve response rates or affect durable remissions.

DTIC is currently the only chemotherapeutic agent approved for use inmetastatic melanoma. The efficacy of dacarbazine in the treatment ofmetastatic melanoma is very dependent on disease site and, according tothe most recent publications and abstracts (Journal of Clinical Oncologyand ASCO annual meeting proceedings, 2004), the actual overall responsesto DTIC are 5.5-6.8%, with responses being short-lived (i.e., three tosix months). There is no evidence that these responses have any effecton the patients' overall survival.

Other drugs investigated for use alone or in combination withdacarbazine, include: alkylating agents and nitrosureas; vincaalkaloids; platinum compounds; hormonal agents; and plant-derived agents(paclitaxel (TAXOL), coumarin). None of these drugs, either alone or incombination with dacarbazine and/or Interferon alpha have been shown tobe any more effective than dacarbazine alone (Cancer Medicine, Ed. 52000; pp. 1849-69) and are considered useful only for symptomaticrelief.

In the medical field there is a pressing need to develop new therapiesfor stage IV malignant melanoma characterized by distant unresectablemetastases.

As above mentioned to date, DTIC is currently the only chemotherapeuticagent approved for use in metastatic melanoma. The actual overallresponses to DTIC are 5.5-6.8%; and there is no evidence that theseresponses have any effect on the patients' overall survival.

To date, the use of thymosin alpha 1 (a) in a dose higher than 1 mg/s.c.in combination with dacarbazine and/or Interferon alpha; for preparing amedicament for the treatment of malignant melanoma on stage IVcharacterized by distant unresectable metastases, was not known in theart.

DESCRIPTION OF THE INVENTION

It has now been found that relatively high doses of thymosin alpha 1 incombination with dacarbazine, and optionally Interferon alpha, areuseful for treating malignant melanoma on stage IV characterized bydistant unresectable metastases, particularly on patients having normalserum level of LDH (lactate dehydrogenase).

For purposes of the present invention, the phrase “relatively highdoses” as it pertains to thymosin alpha 1 shall be understood to meandoses in excess of about 1 mg per parenteral, e.g. subcutaneous,administration.

For purposes of the present invention, the phrase “low serum level ofLDH (lactate dehydrogenase)” shall be understood to mean levels belowabout 460 U/L (normal levels are from 96 to 460 U/L).

It is therefore an object of the present invention to provide a use ofthymosin alpha 1 in a dose higher than 1 mg/day/s.c., in combinationwith dacarbazine, and optionally Interferon alpha, for preparing amedicament for the treatment of malignant melanoma on stage IVcharacterized by distant unresectable metastases;

in which:

thymosin alpha 1 is administered in a dose from 1.1 to 7 mg/day/s.c.;the preferred dose is from 1.6 to 6.4 mg/day/s.c.; the most preferreddose are 1.6; 3.2; and 6.4 mg/day/s.c.;

dacarbazine is administered in a dose from 500 to 1100 mg/m²/day/i.v.;the preferred dose is 800 mg/m²/day/i.v.; and

Interferon alpha is administered in a dose from 2 to 4 MIU/day/s.c.; thepreferred dose is 3 MIU/day/s.c.

In still further aspects of the invention there are provided methods oftreating malignant melanoma in patients requiring the same. In oneembodiment, the method includes administering a combination of thymosinalpha 1 and dacarbazine, and optionally Interferon alpha, to a patientin need thereof. As administered herein, the combination of thymosinalpha 1 and dacarbazine in the amounts described herein providetherapeutic advantages over the administration of either agent alone orprior art combinations of the ingredients in the treatment of melanomas,including malignant melanomas. Those of ordinary skill will appreciatethat although the methods described herein speak of combinations of thetwo primary therapeutic agents, it is contemplated that each of thetherapeutic agents can and preferably will be administered to thepatient separately rather than as part of a single pharmaceutical dosageform or even simultaneously to the patient in need thereof.

It will also be understood that the inventive methods of use andtreatment contemplate administration of the synergistic combinations aspart of treatment protocols as such protocols are understood by those ofordinary skill. Without wishing to be bound by particulars, suchtreatment protocols can call for administration of the combinationsaccording to a schedule which can be repeated, as needed. See, forexample, the 28 day cycle described in Example 1 below. Further cyclesand protocols will be apparent to those of ordinary skill based upon thedescription provided herein and clinical expense, without undueexperimentation. Other protocols for treating malignant melanoma in apatient, include administering a synergistic combination of thymosinalpha 1 and dacarbazine to patient in need thereof, wherein thecombination is administered according to a protocol in which thedacarbanize is administered on day 1 thereof and the thymosin alpha 1 isadministered between about one week and about two weeks thereafter. Analternative protocol for treating malignant melanoma in a patientincludes administering a synergistic combination of thymosin alpha 1,dacarbazine and Interferon alpha to patient in need thereof, wherein thecombination is administered according to a protocol in which thedacarbazine is administered on day 1 thereof, the thymosin alpha 1 isadministered about between one week and about two weeks thereafter andthe Interferon alpha is administered about 10-12 days and optionallyabout 18 days after the dacarbazine is administered.

A still further aspect of the invention includes a kit for treatingmelanomas such as malignant melanoma. The kits include effective amountsof thymosin alpha 1, dacarbazine, and optionally Interferon alpha.

Since the present invention relates in certain embodiments to using acombination of active ingredients wherein the active ingredients may beadministered separately, the invention also relates to combiningseparate pharmaceutical compositions in kit form. That is, a kit iscontemplated wherein the two principal agents, i.e. thymosin alpha 1 anddacarbazine are present, as described above. The kit will preferablyinclude directions for the administration of the separate components.The kit form is particularly advantageous when the separate componentscan be administered in different dosage forms (e.g., oral andparenteral) or are administered at different dosage intervals as willmost commonly be the case herein where the components are administeredon different days.

For purposes of the present invention “effective amount” shall beunderstood to mean an amount which achieves a desired clinical result,i.e. reduction, slowing, remission, etc. or reversal of the malignantmelanoma condition in the patient, i.e. mammal or human.

EXAMPLES

The following examples further illustrates the invention but are notmeant in any way to restrict the effective scope of the invention.

Example 1

Phase II Clinical Trial with Dacarbazine (DTIC) Plus Thymosin Alpha 1(Tα1) with or without Interferon Alpha (IFNα) vs DTIC Plus IFNα in StageIV Melanoma Characterized by Distant Unresectable Metastases

Trial Design phase II, randomized, stratified, open-study testingdifferent doses of Tα1 in association with DTIC and IFNα, as first linetherapy for stage IV melanoma patients characterized by distantunresectable metastases (AJCC; Journal of Clinical Oncology 2001, 19:3635-3648) without brain metastases. The primary study end-point wastumor response, and the following combination composition were used:

-   -   DTIC (800 mg/m²)+IFNα (3 MIU)+Tα1 (1.6 mg) (97 pts);    -   DTIC (800 mg/m²)+IFNα (3 MIU)+Tα1 (3.2 mg) (97 pts);    -   DTIC (800 mg/m²)+Tα1 (3.2 mg) (98 pts);    -   DTIC (800 mg/m²)+IFNα (3 MIU) (94 pts);

During a preliminary analysis on 142 patients surprisingly andunexpectedly it was discovered that a clear dose-response effect wasobserved at the higher doses of Tα1.

The protocol was than amended and the following new group of 97 patientstreated with a higher dose of Tα1 was added:

-   -   DTIC (800 mg/m²)+IFNα α (3 MIU)+Tα1 6.4 mg (97).

The five groups were analyzed independently one another within theso-called “pick the winner” strategy.

Methods: Recycling every 28 days, patients were administered DTIC (800mg/m²) i.v. at day 1, Tα1 (1.6, 3.2 or 6.4 mg) s.c. at days 8-11 and15-18, and IFNα (3 MIU) s.c. at day 11 and 18. Clinical response wasevaluated every two cycles according to RECIST criteria (New Guidelinesto Evaluate the Response to Treatment in Solid Tumors; Journal of theNational Cancer Institute, 2000. 92: 205-216) utilizing a centralreader.

The randomized patients were stratified according to the disease site:M1a, M1b or M1c level.

1) Patients with cutaneous, subcutaneous and/or limphnodal metastaseswith normal serum LDH value (from 96 to 460 U/L) were classified as M1a.

2) Patients with lung metastases and normal serum LDH value wereclassified as M1b.

3) Patients with other visceral metastases and/or with serum LDH valueout of normal range were classified as M1c.

M1b patients notoriously have worse prognosis than M1a patients whilethe M1c patients have the worst prognosis.

It has to be emphasized that, at the time of the preliminary analysis,the distribution of the patients population among strata was as follows:16% M1a, 25% M1b, 59% M1c. Therefore, this population is very similar tothe one for which only 5% of DTIC efficacy has been found in literaturein the most recent publications: Journal of Clinical Oncology, 2004, 22:1118-1125; Journal of Clinical Oncology, 2004 ASCO Annual MeetingProceedings (Post-Meeting Edition), 22, No 14S (July 15 Supplement):7543); Journal of Clinical Oncology, 2004 ASCO Annual MeetingProceedings (Post-Meeting Edition), 22, No 14S (July 15 Supplement):7505; Journal of Clinical Oncology, 2004 ASCO Annual Meeting Proceedings(Post-Meeting Edition), 22, No 14S (July 15 Supplement): 7508). Thisdistribution was maintained the same at the end of the recruitment.

Furthermore, according to the RECIST criteria (the most widely usedcriteria to evaluate response to the treatment in solid tumors) overallresponses (OR) have to be confirmed after at least 4 weeks: if notconfirmed, patient is considered as being in a stable disease (SD)condition.

Patients were treated with up to 6 cycles, unless one of the followingthree conditions appeared:

1) Development of any serious adverse event (SAE), unexpected worseningof the patient's basal conditions which would make participation in thetrial inappropriate;

2) Progression of disease;

3) Withdrawal by patient of the consent to participate to the trial.

In all these cases, the patients were withdrawn from the study.

Patients who, at the end of the 6 cycles, were in SD, Partial Response(PR) or Complete Response (CR), could be treated further on, accordingto the Physician's opinion, until a maximum of 24 cycles.

The results of a clinical trial, reported in the following Tables1/A-5/A relate to patients treated without considering their LDH serumlevels.

The results obtained, reported in the following Tables 1/B-5/B relatesto patients with a serum level of LDH between 96 to 460 U/L (this is asub population of the patients treated/present in Tables 1/A-5/A.

TABLE 1/A Dacarbazine (800 mg/m²) + IFNα Interferon alpha (3 MIU)(control group) RESULTS Patients Evaluated 94 Complete Response Rate 0Partial Response Rate 5 Complete Response Rate + 5 (5.3%) PartialResponse Rate

TABLE 1/B Dacarbazine (800 mg/m²) + IFNα Interferon alpha (3 MIU)(control group) RESULTS EXCLUDING ELEVATED LDH Patients Evaluated 62Complete Response Rate 0 Partial Response Rate 3 Complete ResponseRate + 3 (4.8%) Partial Response Rate

TABLE 2/A Dacarbazine (800 mg/m²) + IFNα Interferon alpha (3 MIU) +Thymosin alpha 1 (1.6 mg) RESULTS # of Patients Evaluated 97 CompleteResponse Rate 2 Partial Response Rate −5 Complete Response Rate + 7(7.2%) Partial Response Rate

TABLE 2/B Dacarbazine (800 mg/m²) + IFNα Interferon alpha (3 MIU) +Thymosin alpha 1 (1.6 mg) RESULTS EXCLUDING ELEVATED LDH # of PatientsEvaluated 64 Complete Response Rate 2 Partial Response Rate 5 CompleteResponse Rate + 7 (10.9%) Partial Response Rate

TABLE 3/A Dacarbazine (800 mg/m²) + Interferon alpha (3 MIU) + Thymosinalpha 1 (3.2 mg) RESULTS # of Patients Evaluated 97 Complete ResponseRate 2 Partial Response Rate 8 Complete Response Rate + 10 (10.3%)Partial Response Rate

TABLE 3/B Dacarbazine (800 mg/m²) + Interferon alpha (3 MIU) + Thymosinalpha 1 (3.2 mg) RESULTS EXCLUDING ELEVATED LDH # of Patients Evaluated58 Complete Response Rate 2 Partial Response Rate 6 Complete ResponseRate + 8 (13.8%) Partial Response Rate

TABLE 4/A Dacarbazine (800 mg/m²) + Interferon alpha (3 MIU) + Thymosinalpha 1 (6.4 mg) RESULTS # of Patients Evaluated 97 Complete ResponseRate 2 Partial Response Rate 4 Complete Response Rate + Partial 6 (6.2%)Response Rate

TABLE 4/B Dacarbazine (800 mg/m²) + Interferon alpha (3 MIU) + Thymosinalpha 1 (6.4 mg) RESULTS EXCLUDING ELEVATED LDH # of Patients Evaluated62 Complete Response Rate 2 Partial Response Rate 4 Complete ResponseRate + 6 (9.7%) Partial Response Rate

TABLE 5/A Dacarbazine (800 mg/m²) + Thymosin alpha 1 (3.2 mg) RESULTS #of Patients Evaluated 98 Complete Response Rate 2 Partial Response Rate11 Complete Response Rate + 13 (13.3%) Partial Response Rate (%)

TABLE 5/B Dacarbazine (800 mg/m²) + Thymosin alpha 1 (3.2 mg) RESULTSEXCLUDING ELEVATED LDH # of Patients Evaluated 59 Complete Response Rate2 Partial Response Rate 9 Complete Response Rate + 11 (18.6%) PartialResponse Rate (%)

The results reported in Tables 1/A-5/A surprisingly, and unexpectedly,show that the combination according to the invention is therapeuticallymore active than DITIC in combination with IFNα.

In fact the control group shows a response of 5.3% (Table 1/A) while theother groups show a response from 6.2 (table 4/A) to 13.3% (table 5/A).

These results were confirmed in the sub population composed of patientswithout an elevated baseline level of serum LDH. In fact the controlgroup shows a response of 4.8% (Table 1/B while the other groups show aresponse from 9.7 (table 4/B) to 18.6% (table 5/B).

For a pathology in which: (a) DTIC is the only chemotherapeutic agentapproved, (b) the actual overall responses to DTIC are 5.5-6.8%, and (c)there is no evidence that these responses have any effect on thepatients' overall survival; the results above reported have shown areally surprisingly unexpected therapeutic effect.

The daily dose of the active ingredients to be administered will depend,according to the judgement of the primary care physician, on thesubject's weight, age or general condition.

Thymosin alpha 1, dacarbazine and Interferon alpha are well known activeingredients used in the medical field.

1. A method of treating stage IV malignant melanoma classified as M1a,M1b or M1c, consisting of administering: thymosin alpha 1 in a dose of6.4 mg/day/s.c., in combination with an effective amount of dacarbazine,to a human with stage IV malignant melanoma classified as M1a, M1b orM1c.
 2. Method according to claim 1, in which dacarbazine isadministered in a dose from 500 to 1100 mg/m²/day/i.v.
 3. Methodaccording to claim 1, in which dacarbazine is administered in a dose of800 mg/m²/day/i.v.
 4. Method according to claim 1, in which the human tobe treated has a normal serum level of LDH.
 5. A method of treatingmalignant melanoma in a human patient, consisting of administering asynergistic combination of thymosin alpha 1 and dacarbazine to a patientwith stage IV malignant melanoma classified as M1a, M1b or M1c, whereinsaid combination is administered according to a protocol in which thedacarbazine is administered in a dose of 800 mg/m²/day/i.v., on day 1thereof and the thymosin alpha 1 is administered in a dose of 6.4mg/day/s.c. about one week and about two weeks thereafter.
 6. Methodaccording to claim 5, in which the human to be treated has a normalserum level of LDH.